Bottom Line:
In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1-/-) SMCs.Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFbeta signaling, as indicated by high levels of nuclear phospho-Smad2.TGFbeta activation alone is not sufficient for the expression of the Marfan-like vascular phenotype.

Methods and principal findings: In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1-/-) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor beta (TGFbeta) signaling are present in smooth muscle-specific LRP1 knockout (smLRP1-/-) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRbeta in atherogenesis, we generated a strain of smLRP1-/- mice in which tyrosine 739/750 of the PDGFRbeta had been mutated to phenylalanines (PDGFRbeta F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP1-/- animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFbeta signaling, as indicated by high levels of nuclear phospho-Smad2.

Conclusions and significance: Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRbeta-dependent activation of PI3K. TGFbeta activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRbeta is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome.

pone-0006922-g005: Primary SMC Migration.(A) Transwell migration assay. 30,000 SMCs of the indicated genotypes were added to the top compartment of a Boyden chamber. After 6 hours of incubation, the transwell membrane was fixed and stained with hematoxylin. Cells that had migrated through the holes on the membrane to the bottom face were counted. Scale bar, 50 µm. (B) Scratch assay. 300,000 SMCs of the indicated genotypes were seeded into 60 mm culture dishes in a medium containing 10 µg/ml mitomicin C to prevent cell proliferation and allowed to adhere overnight. The next day, part of the dish was denuded by scratching along a straight line (indicated by a black line behind). Cells were then allowed to migrate into the denuded area for 24 hours prior to fixation and quantification. Scale bar, 50 µm. (C, D) Statistical analyses of the Transwell and Scratch migration assays. Results are represented as mean±SD. *** p<0.001. (E) PDGF-BB chemotaxis assay. 10,000 SMCs of the indicated genotypes were added to the top compartment of the Boyden chamber. 10 ng/ml PDGF-BB was added to the lower chamber of the well. After 6 hours of incubation, the transwell membrane was fixed and stained with hematoxylin. The cells that had migrated through the holes on the membrane to the bottom face were counted. Scale bar, 50 µm. (F) Statistical analysis of the PDGF-BB chemotaxis assay. Results are represented as mean±SD. ** p<0.01, *** p<0.001. n = 5 for all assays.

Mentions:
Our in vivo experiments showed disarranged SMCs in the medial layer of the aorta (Figure 1m, 3h, 3k) and disrupted elastic laminas when LRP1 was deficient in the SMCs (Figure 1s, 3t, 3w). To investigate if these phenomena are caused by abnormal migration due to the absence of LRP1, we performed two different kinds of in vitro migration assays. Compared with wild type cells, SMCs lacking LRP1 showed markedly increased migratory activity, both in a Boyden chamber transmigration assay (Figure 5A, C) and in the commonly used scratch assay, in which the migration of the cells into a denuded area of a tissue culture dish is quantified (Figure 5B, D). Cell migration was significantly reduced in those cells containing the PDGFRβ F2/F2 mutation (Figure 5A–D), and this correlated with the improved architecture of the elastic layers in the aortic wall of smLRP−/−; LDLR−/−; PDGFRβ F2/F2 mice (Figure 3u, x). These findings thus confirm that SMC migration is regulated by LRP1 through the PDGFRβ-dependent PI3K pathway.

pone-0006922-g005: Primary SMC Migration.(A) Transwell migration assay. 30,000 SMCs of the indicated genotypes were added to the top compartment of a Boyden chamber. After 6 hours of incubation, the transwell membrane was fixed and stained with hematoxylin. Cells that had migrated through the holes on the membrane to the bottom face were counted. Scale bar, 50 µm. (B) Scratch assay. 300,000 SMCs of the indicated genotypes were seeded into 60 mm culture dishes in a medium containing 10 µg/ml mitomicin C to prevent cell proliferation and allowed to adhere overnight. The next day, part of the dish was denuded by scratching along a straight line (indicated by a black line behind). Cells were then allowed to migrate into the denuded area for 24 hours prior to fixation and quantification. Scale bar, 50 µm. (C, D) Statistical analyses of the Transwell and Scratch migration assays. Results are represented as mean±SD. *** p<0.001. (E) PDGF-BB chemotaxis assay. 10,000 SMCs of the indicated genotypes were added to the top compartment of the Boyden chamber. 10 ng/ml PDGF-BB was added to the lower chamber of the well. After 6 hours of incubation, the transwell membrane was fixed and stained with hematoxylin. The cells that had migrated through the holes on the membrane to the bottom face were counted. Scale bar, 50 µm. (F) Statistical analysis of the PDGF-BB chemotaxis assay. Results are represented as mean±SD. ** p<0.01, *** p<0.001. n = 5 for all assays.

Mentions:
Our in vivo experiments showed disarranged SMCs in the medial layer of the aorta (Figure 1m, 3h, 3k) and disrupted elastic laminas when LRP1 was deficient in the SMCs (Figure 1s, 3t, 3w). To investigate if these phenomena are caused by abnormal migration due to the absence of LRP1, we performed two different kinds of in vitro migration assays. Compared with wild type cells, SMCs lacking LRP1 showed markedly increased migratory activity, both in a Boyden chamber transmigration assay (Figure 5A, C) and in the commonly used scratch assay, in which the migration of the cells into a denuded area of a tissue culture dish is quantified (Figure 5B, D). Cell migration was significantly reduced in those cells containing the PDGFRβ F2/F2 mutation (Figure 5A–D), and this correlated with the improved architecture of the elastic layers in the aortic wall of smLRP−/−; LDLR−/−; PDGFRβ F2/F2 mice (Figure 3u, x). These findings thus confirm that SMC migration is regulated by LRP1 through the PDGFRβ-dependent PI3K pathway.

Bottom Line:
In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1-/-) SMCs.Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFbeta signaling, as indicated by high levels of nuclear phospho-Smad2.TGFbeta activation alone is not sufficient for the expression of the Marfan-like vascular phenotype.

Methods and principal findings: In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1-/-) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor beta (TGFbeta) signaling are present in smooth muscle-specific LRP1 knockout (smLRP1-/-) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRbeta in atherogenesis, we generated a strain of smLRP1-/- mice in which tyrosine 739/750 of the PDGFRbeta had been mutated to phenylalanines (PDGFRbeta F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP1-/- animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFbeta signaling, as indicated by high levels of nuclear phospho-Smad2.

Conclusions and significance: Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRbeta-dependent activation of PI3K. TGFbeta activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRbeta is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome.